The kidney was the first organ whose function was approximated by a machine and a filter device, and was also the first organ which was successfully transplanted. However, the lack of widespread availability of transplantable organs reserved the rights for transplantation only to patients with chronic renal failure.
The development of acute renal failure (ARF) in a hospitalized patient results in a 5-fold to 8-fold higher risk of death. Although hemodialysis, hemofiltration and peritoneal dialysis treatment with its small solute and fluid clearance function has prevented death from hyperkalemia, volume overload and uremic complications, such as pericarditis, patients with ARF still have mortality rates exceeding 50. It is not a complete renal replacement therapy because it only provides filtration function and does not replace the hemostatic, regulatory, metabolic, and endocrine function. Patients with end stage renal disease on dialysis continue to have major medical, social and economic problems.
Therefore, it is important to invest into the improvement and the development of alternatives to the existing therapies. “Bioartificial” or “hybrid” organs are a promising and realistic alternative to the presently available therapies for the treatment of renal failure.
Acute renal failure as a secondary effect of ischemic and/or nephrotoxic causes arises from acute tubular necrosis (ATN), predominantly to renal proximal tubule cells. A support of these tubular cells and thus a naturally replacement in function during the first time of ATN could provide almost full renal replacement therapy in conjunction with hemofiltration. Additionally with such a support the other main functions of these cells, their metabolic activity such as ammoniagenesis and glutathione reclamation, endocrine activity such as vitamin D3 activation and cytokine homeostasis, may provide additional physiologic replacement activities that have the potential to change the current natural history of this disease process.
The knowledge of the cellular and molecular basis of organ function and diseases will be transferred in the next years into new therapeutic approaches. Central to these are the developing fields of gene therapy, cell therapy and tissue engineering. These new potentialities are based on the ability to expand stem or progenitor cells in tissue culture to perform different tasks and to introduce these cells into the patient either in extra-corporeal circuits or as implantable constructs.
Cell therapy is a new and exciting approach to the treatment off acute and chronic diseases. The potential success of such new ways lies in the growing appreciation that most diseases are not due to the lack or excess of single events but develop due to alterations in the complex interactions of a variety of cell products. In addition, cells organs and tissue need a specific individualized therapy that responds to the pathophysiological conditions. This form of treatment is dependent on cell and tissue culture methodology to isolate, expand and supply specific cells which may replace important processes that are deranged or lost in various disease states. Recent approaches are, for example, placing cells on flat sheet membranes, into hollow fibers or encapsulating substances, and to develop bioreactors for the delivery of active cells to a patient.
The kidney is probably the most challenging organ to reconstruct by tissue engineering techniques, because of its complex structure and function. The nephron is the basic structural and functional unit of the kidney. Its chief function is to regulate water and soluble substances by filtering the blood, reabsorbing what is needed and excreting the rest as urine. Nephrons eliminate wastes from the body, regulate blood volume and pressure, control levels of electrolytes and metabolites, and regulate blood pH. Its functions are vital to life and are regulated by the endocrine system by hormones such as antidiuretic hormone, aldosterone, and parathyroid hormone. Each nephron is composed of an initial filtering component (the renal corpuscle) and a tubule specialized for reabsorption and secretion (the renal tubule). The renal corpuscle filters out large solutes from the blood, delivering water and small solutes to the renal tubule for modification.
The most distinctive characteristic of the proximal tubule is its “brush border”. The luminal surface of the epithelial cells of this segment of the nephron is covered with densely packed microvilli forming a border readily visible under the light microscope. The microvilli greatly increase the luminal surface area of the cells, presumably facilitating their resorptive function. The cytoplasm of the cells is densely packed with mitochondria in keeping with the energetic requirements of the cells resorptive activity. Fluid in the filtrate entering the proximal convoluted tubule is reabsorbed into the peritubular capillaries, including approximately two-thirds of the filtered salt and water and all filtered organic solutes (primarily glucose and amino acids). This is driven by sodium transport from the lumen into the blood by the Na+/K+ ATPase in the basolateral membrane of the epithelial cells. Much of the mass movement of water and solutes occurs in between the cells through the tight junctions, which in this case are not selective.
The distal convoluted tubule is similar to the proximal convoluted tubule in structure and function. Cells lining the tubule have numerous mitochondria, enabling active transport to take place by the energy supplied by ATP. Much of the ion transport taking place in the distal convoluted tubule is regulated by the endocrine system. In the presence of parathyroid hormone, the distal convoluted tubule reabsorbs more calcium and excretes more phosphate. When aldosterone is present, more sodium is reabsorbed and more potassium excreted. Atrial natriuretic peptide causes the distal convoluted tubule to excrete more sodium. In addition, the tubule also secretes hydrogen and ammonium to regulate pH. After traveling the length of the distal convoluted tubule, only 3% of water remains, and the remaining salt content is negligible.
So far, human proximal and distal tubule cells, which could be used for clinical approaches to the above described problem, could not successfully be isolated, characterized and kept in culture in their highly differentiated state (for a review on the development of artificial kidneys see Fissell: Development towards an artificial kidney. Expert Rev. Med. Devices 2006, 3(2), 155-165). Such cells, however, form the basis for the development of a hybrid human kidney.
Therefore, it was the aim of the present invention to develop a hybrid bioartificial kidney equivalent which is able to replace the main functions, both metabolic and endocrine, of a healthy human renal tubular system.
In 1987, Aebischer et al. first reported the concept of a bioartificial kidney in that tubular epithelial cells formed confluent monolayers on the outer surfaces of a hollow fiber membrane module and had transport ability for water and solutes across the cells and membrane (Aebischer et al.: Renal epithelial cells grown on semipermeable processor. Trans. Am. Soc. Artif. Intern. Organs 1987, 33, 96-102; Aebischer et al.: The bioartificial kidney: progress toward an ultrafiltration device with renal epithelial cells processing: Life Support Sys. 1987, 5, 159-68). Results achieved by a group around H. David Humes (Humes et al.: Replacement of renal function in uremic animals with a tissue-engineered kidney. Nature Biotechnology 17, 451-455, 1999; Humes et al.: Tissue engineering of a bioartificial renal tubule assist device: In vitro transport and metabolic characteristics. Kidney International 55, 2502-2514, 1999; Humes et al.: The bioartificial kidney in the treatment of acute renal failure. Kidney International 61, S121-S125, 2002; U.S. Pat. No. 6,942,879 B2) show that it is in principle possible to assemble a kind of artificial kidney in the form of an extracorporeal filtration and reclamation circuit which incorporates living epithelial cells of the kidney proximal tubule into its design.
This design is based on an extracorporeal device using a standard hemofiltration cartridge containing approximately 109 renal tubule cells grown as confluent monolayer along the inner surface of the fibers which were coated with an extracellular matrix. The non-biodegradability and the pore size of the hollow fibers allow the membranes to act as scaffolds for the cells and as an immunoprotective barrier. In vitro studies of this renal tubule assist device (RAD) have shown that the cells retain their functionality, referred active transport properties, metabolic activities and important endocrinal processes. The combination of synthetic hemofiltration cartridge in series with this RAD in a second step formulates a tissue-engineered bioartificial kidney which can be used for a more complete renal replacement therapy. In brief, blood enters the fibers of the hemofilter where ultrafiltrate is formed and delivered into the fibers of the tubular lumen in the horizontally oriented RAD. Said ultrafiltrate can be called urine. The filtered blood exiting the hemofilter enters the RAD through the extracapillary space port and disperses among the fibers. At the end of the RAD ultrafiltrate and filtered blood will be collected and the blood is returned to the patient. Heparin is delivered continuously into the blood before entering the RAD to diminish clotting within the device.
The approach of Humes has certain drawbacks, as only proximal tubule cells are used in the design. All other functionally important parts of the tubule system of the kidney, such as the early distal segment, are not available. As described before, the kidney distal segment regulates, for example, the exchange of Na+ for K+ under aldosterone regulation, reabsorption of bicarbonate ion, secretion of hydrogen ion, and conversion of ammonia to ammonium ion. The kidney distal segment is also the place for the formation of other important molecules, such as EGF, cytokines etc. Further, the luminal surfaces of the membranes used by Humes et al. have to be coated e.g. with pronectin-L, a recombinant protein that promotes cell adhesion. It would be preferable, however, to use human proximal and distal epithelial tubule cells instead of animal cells, and to minimize the use of additional, extracorporeal substances in the system, which might promote adverse effects.
In another approach, described e.g. by Saito (Saito A.: Research into the Development of a wearable Bioartificial Kidney with a Continuous Hemofilter and a Bioartificial Tubule Device Using Tubular Epithelial Cells. Artificial Organs 2004, 28(1), 58-63), LLC-PK1 cells (porcine kidney cells) and MDCK cells (canine kidney cells) were seeded inside polysulfone or cellulose acetate hollow fibers. Again this tubule device makes use of non-human proximal tubular epithelial cells only. The membranes used were coated with extracellular matrices, and the tubular epithelial cells were transfected with functional genes, such as the rat aquaporin-1 gene (Saito et al.: Present Status and Perspective of the Development of a Bioartificial Kidney for Chronic Renal Failure Patients. 2006, Therapeutic Apheresis and Dialysis 10(4), 342-347).
In contrast to the designs of the prior art, the present invention devises an improved hybrid bioartificial kidney in that a proximal and distal unit are combined, thereby further regulating in the distal unit reabsorption of water, sodium chloride and calcium. Further, due to the use of a specifically designed hollow fiber membrane, the culturing of the renal tubular cells can be achieved without additionally coating the membrane with extracorporeal matrices, which is another step closer to the development of an artificial kidney.